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Logan et al. BMC Genomics 2014, 15:828
http://www.biomedcentral.com/1471-2164/15/828
METHODOLOGY ARTICLE
Open Access
A universal protocol to generate consensus level
genome sequences for foot-and-mouth disease
virus and other positive-sense polyadenylated
RNA viruses using the Illumina MiSeq
Grace Logan†, Graham L Freimanis*†, David J King, Begoña Valdazo-González, Katarzyna Bachanek-Bankowska,
Nicholas D Sanderson, Nick J Knowles, Donald P King and Eleanor M Cottam
Abstract
Background: Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new
approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and
veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered
during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We
describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina
MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and
circumvents both the use of PCR and the requirement for large amounts of initial template.
Results: The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic
in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this
protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell
culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV)
were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads
was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates
and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately
flanking the poly(C) region.
Conclusions: We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses.
This method works successfully from a limited quantity of starting material and eliminates the requirement for
genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a
routine high-throughput diagnostic environment.
Keywords: Next generation sequencing, Whole genome sequencing, Foot-and-mouth disease virus, Genome, RNA,
virus, FMDV
* Correspondence: [email protected]
†
Equal contributors
The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United
Kingdom
© 2014 Logan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Logan et al. BMC Genomics 2014, 15:828
http://www.biomedcentral.com/1471-2164/15/828
Background
Foot-and-mouth disease (FMD) has been associated with
severe productivity losses in cloven-hoofed animals characterised by vesicular lesions of the feet, tongue, snout and
teats as well as fever and lameness [1]. The disease has a
serious impact upon food security, rural income and
significant economic consequences for any country harbouring the virus [2]. An integral part of any viral disease
control strategy is the epidemiological tracing of virus
transmission together with conventional field investigations. For RNA viruses with high evolutionary rates, this is
routinely achieved with the application of molecular and
phylogenetic methods [3-5] one example being the global
tracing of foot-and-mouth disease virus (FMDV) [6].
Next-generation sequencing platforms offer much promise
as rapid, cost-effective, and high-throughput methods for
the generation of viral genome sequences. Recovering
whole genome consensus level sequences of viruses provides important information for outbreak epidemiology
and pathogen identification [7-10].
The positive-sense single-stranded RNA genome of
FMDV is comprised of a single long open reading frame.
This encodes a polyprotein which is flanked by 5′ and 3′
untranslated regions of approximately 1200 nt and 95 nt,
respectively, terminating in a poly (A) tail. The 5′ UTR
contains highly structured RNA which is involved in both
replication and translation. Approximately 300–370 nt
from the 5′ end of the genome lies a homopolymeric
cytidylic acid [poly(C)] tract of ~100-170 nt [11]. The
genome sequence upstream of the poly(C) tract is known
as the S fragment and that downstream as the L fragment.
Previously, tracing and monitoring of the trans-boundary
movements of FMDV has been successfully achieved using
consensus sequences of the VP1 region [12-14]. However,
over shorter epidemic time scales, where viral populations
have not substantially diverged, VP1 sequencing cannot
provide the required resolution to discriminate between
viruses in field samples collected from neighbouring farms
within outbreak clusters. At this scale, WGS at the consensus level has proven to be a powerful tool for the reconstruction of transmission trees [15].
Previous strategies for viral WGS include PCR and
Sanger sequencing methods or microarray approaches
[15,16]. Commonly, these processes have limited throughput and are both resource and labour-intensive with biased
outputs that may not reflect the true diversity within samples [17,18]. Furthermore, such methodologies have been
subject to errors incumbent within the nature of the protocol i.e. those protocols reliant upon DNA amplification
generate biased datasets from which it is difficult to make
firm conclusions [19]. Such strategies have also been
dependent upon a priori knowledge of virus sequences for
primer design and are limited by potential inter and intrasample sequence variation [20].
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This study describes the optimisation of a robust, highthroughput protocol for WGS of all seven serotypes of
FMDV excluding the 5′ genomic termini and poly(C)
tract. It does not use PCR amplification prior to the
sequencing steps and overcomes the requirement for large
starting quantities of template nucleic acid, which has previously limited the suitability of some NGS technologies
for processing viral field isolates [21-23]. This protocol,
with minor changes, was also applied to other polyadenylated RNA viruses.
Results
Protocol accuracy: calculation of minimum coverage
required for accurate consensus
Next-generation sequencing analysis provided large numbers of short read sequences that were assembled and
aligned in order to determine a consensus sequence. To
define how much redundancy was required for accurate reconstruction of consensus level sequences, we determined
the minimum read coverage required to obtain a robust
consensus from the protocol described. Analysis was completed on all FMDV type O samples with sufficient coverage (Figure 1). From this a mean was calculated showing a
minimum coverage of 22 reads was required to obtain an
accurate consensus sequence in this instance.
Figure 1 Read coverage required to obtain an accurate
consensus sequence. The consensus sequence resulting from
varying levels of coverage was assessed for accuracy. Isolates O/
UKG/1450/2001 (blue), O/UKG/1558/2001 (green), O/UKG/1734/2001
(purple), O/UKG/4998/2001 (orange) and O/UKG/14597/2001 (red)
alongside the type O exemplar from the serotype panel (black) were
analysed. Points on the graph represent a comparison of the
identities (scored on the y axis) of a consensus made with total
reads and a consensus made with limited read coverage (detailed
on the x axis). On average, an identity score of one was maintained
up to (and including) a coverage limit of 22 reads. Below this level
of coverage, the accuracy of the identities of the compared
consensus sequences decreased i.e. consensus sequences made
with a depth of 22x reads were identical to the consensus.
Sequences created with less than 22x coverage depth were not
identical, and therefore considered less accurate.
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Analytical sensitivity of WGS protocol: consensus
sequence was obtained to 1 × 107 virus genome copies
Validation of protocol on field samples of FMDV and
reproducibility
The protocol workflow (See Materials and Methods)
was optimised and tested using a single FMDV O/
UKG/35/2001 isolate. Initially, the sensitivity of the
protocol in the presence of gDNA (i.e. no rDNase1
treatment) was tested against viral dilutions spanning
1 × 108, 1 × 107 and 1 × 106 RNA copies/μl. The total
number of Illumina reads in all five samples ranged
between 2.5 × 106 and 1.2 × 106 (Table 1). Consensus
genome sequences (8176 nucleotides in length) were
created from alignments of these reads at each dilution. A decreasing percentage of viral reads correlated
with decreasing viral load (17.94%, 14.41%, 1.83%,
0.05% and 0.01% respectively). Consensus sequences
were found to be identical in all cases both between individual samples and the reference sequence (data not
shown). For this isolate, whole genome sequence was
attained (excluding the 5′ termini) for 1 × 108 and 1 ×
107 genomes copies/μl, however, below this level,
coverage was incomplete. Coverage was increased in
regions adjacent to primer binding sites and was lowest
in the S-fragment (genome positions nt 1–376),
notably in regions immediately adjacent to the poly(C)
tract. The 3’ genomic termini were obtained in the cell
culture neat virus sample (1 × 108 copies/μl) with only
2 bases missing at the 5′ termini. In order to gain
accurate consensus our analysis shows that for type O
we needed a minimum viral read depth of 22. By this
criterion accurate consensus sequences were generated
for >98.1% of the genome, down to 1 × 107 copies/μl.
Below this threshold (i.e. <1 × 107 copies/μl) we observed a rapid drop-off in the coverage depth of genome sequences with average coverage across the
genome dropping from 639 (1 × 107) to 18 (1 × 106)
(Table 1). Furthermore both genomic termini, notably
the 5′ end, were also lost with decreasing viral load.
Five field samples submitted to the UK FMD National
Reference Laboratory (Pirbright, UK) during the UK 2001
outbreak were tested using the sequencing protocol for
UKG specific viruses as described above. Virus load in all
samples was quantified by real-time RT-qPCR (Table 1).
Four of five samples (O/UKG/1450/2001, O/UKG/1558/
2001, O/UKG/1734/2001 and O/UKG/14597/2001) contained between 1.8 × 108 – 5.0 × 108 copies/μl. The
remaining sample (O/UKG/4998/2001) was of lower viral
loads with 1.01 × 107 copies/μl, respectively. The number
of viral reads per sample varied between 1 × 106 (sample
O/UKG/1450/2001) and 1 × 104 (O/UKG/4998/2001),
potentially reflecting differences in viral load. Reads were
trimmed and aligned to a reference sequence FMDV O/
UKG/35/2001 (AJ539141). All samples exhibited increased
coverage at primer specific sites (Figure 2) and decreased
coverage at the sites adjacent to the FMDV poly(C) tract
and at the 5′ termini of the S fragment. Samples with viral
load >1 × 108 copies/μl exhibited >69% of reads aligning
to the reference template. The sample with the lowest
viral load, O/UKG/4998/2001, resulted in 67.5% of reads
aligning to the template. Complete genome sequences
(excluding genomic termini) were obtained for all samples.
Isolate O/UKG/1450/2001, which exhibited the highest
viral load and total numbers of reads, generated a coverage
depth >22 across 99.72% of the genome.
For the five samples that generated a whole genome
sequence, the coverage across the L fragment was even,
peaking in regions of reverse transcription primer binding
(Figure 2). All genome sequences have been submitted to
GenBank (KM257061-KM257065). To evaluate reproducibility, one isolate (O/UKG/35/2001) was sequenced 15
separate times. Analysis was completed on each of these
15 repeats and no changes in the consensus sequence produced were observed.
gDNA depletion increases proportion of reads attributed
to virus genome
Application to cell culture negative FMDV
We investigated the impact of genomic DNA (gDNA)
depletion by rDNase1 treatment upon the final library
complexity. Removal of gDNA was confirmed by Qubit
measurement before and after treatment (data not
shown). Although the majority of DNA in the sample
was eliminated it should be noted that some residual
DNA remained in the sample. Samples that had not
been subjected to rDNase1 treatment contained
increased total number of reads, compared to samples
that had been treated with rDNase1 (average: 1.9 × 106
vs. 3.8 × 105 reads, respectively). However, a higher
percentage of reads aligned with the reference template
for gDNA depleted samples compared to untreated
samples (Table 1).
A diagnostic virus sample O/ISR/2/2013, submitted to the
WRLFMD in 2013, was sequenced using the whole genome
sequencing protocol. The virus could not be isolated in cell
culture, but FMDV RNA was detected with diagnostic realtime reverse transcription-quantitative PCR (RT-qPCR) and
quantified as 4.5 × 106 copies/μl (Table 1). The majority of
the genome sequence was generated [(94.10%), with an
average coverage depth of 18] with several gaps evident
across the genome length (Additional file 1: Figure S1).
Pan-FMDV application of WGS protocol
After validation with FMDV UKG field samples the protocol was used to determine whole genome sequences for a
panel of RNA viruses representing each of the seven
FMDV serotypes (Figure 3). In order to optimise the
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Table 1 Library complexity of all samples run whilst optimising the protocol for whole genome sequencing
Sample ID
Serotype
Dnase
treatment
Viral load
(cp/μl)
Total no.
reads
Total viral
reads
Percentage
viral reads
Mean coverage
across genome
Percentage consensus
> depth 22
UKG/35/
2001
FMDV-O
N
4.47 × 108
1.21 × 106
2.17 × 105
17.94
3965
99.28
UKG/35/
2001
FMDV-O
N
1.65 × 108
1.77 × 106
2.55 × 105
14.41
4641
99.3
UKG/35/
2001
FMDV-O
N
3.98 × 107
1.92 × 106
3.51 × 104
1.83
639
98.12
UKG/35/
2001
FMDV-O
N
7.94 × 106
2.08 × 106
1 × 103
0.05
18
38.35
UKG/35/
2001
FMDV-O
N
1.35 × 106
2.47 × 106
1.75 × 102
0.01
3
0
UKG/35/
2001
FMDV-O
Y
4.47 × 108
4.63 × 105
1.19 × 105
25.83
2178
99.36
UKG/35/
2001
FMDV-O
Y
1.65 × 108
1.76 × 105
4.11 × 104
23.37
743
98.29
UKG/35/
2001
FMDV-O
Y
3.98 × 107
3.29 × 105
8.29 × 103
2.52
149
93.71
UKG/35/
2001
FMDV-O
Y
7.94 × 106
4.62 × 105
1.07 × 103
0.23
19
35.71
UKG/35/
2001
FMDV-O
Y
1.35 × 106
3.73 × 105
1.11 × 102
0.03
2
0
UKG/1734/
2001
FMDV-O
Y
2.89 × 108
5.14 × 105
4.12 × 105
80.12
6961
99.46
UKG/1450/
2001
FMDV-O
Y
4.95 × 108
1.23 × 106
1.10 × 106
88.97
18362
99.72
UKG/14597/
2001
FMDV-O
Y
1.77 × 108
2.94 × 105
2.03 × 105
69.02
3557
97.67
UKG/1558/
2001
FMDV-O
Y
4.39 × 108
6.11 × 105
5.27 × 105
86.29
9391
99.68
UKG/4998/
2001
FMDV-O
Y
1.01 × 107
2.97 × 104
2.01 × 104
67.49
352
80.55
TUR/11/
2013
FMDV-O
Y
2.22 × 109
1.29 × 106
8.22 × 105
63.92
14848
99.57
TUR/12/
2013
FMDV-A
Y
7.06 × 108
1.18 × 106
5.51 × 105
46.49
10011
-
KEN/1/2004
FMDV-C
Y
4.41 × 108
1.17 × 106
4.61 × 105
39.45
8049
-
9
6
5
TUR/13/
2013
FMDVAsia 1
Y
2.03 × 10
1.69 × 10
9.04 × 10
53.61
10241
-
TAN/22/
2012
FMDVSAT 1
Y
1.14 × 109
1.43 × 106
7.26 × 105
50.9
13185
-
TAN/5/2012
FMDVSAT 2
Y
1.35 × 109
1.18 × 106
5.35 × 105
45.48
9724
-
ZIM/6/91
FMDVSAT 3
Y
1.47 × 109
2.70 × 106
1.36 × 105
50.21
2453
-
VR-129B
EMCV-1
Y
-
2.63 × 106
2.12 × 106
80.34
31208
-
-
4
2.68 × 104
70.98
409
-
5
4
D1305-03
ERAV-1
Y
3.78 × 10
B1-34
VESV-B34
Y
-
4.77 × 10
6.84 × 10
14.34
1112
-
ISR/2/2013
FMDV-O
Y
4.50 × 106
16 × 104
1.05 × 103
6.53
18
-
N = no; Y = yes; cp = copies.
Different factors of library complexity including total number of reads, number of viral reads, coverage and mean coverage depth across the genome (percentage
consensus depth indicates areas in which depth is over 22).
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consensus against which all reads were aligned. All viruses
gave similar depth of coverage (approx. 1 × 104) and exhibited comparable library complexity with the exception of
SAT 3 whose depth of coverage was reduced (average
coverage: 1 × 103) (Table 1). The 5′ genomic termini was
also missing from all panel viruses ranging from 9 bases of
A and Asia1 to 15, 17, 22 and 27 for SAT 2, SAT 1, SAT 3
and O respectively (accession numbers KM268895-901).
Application to non-FMDV RNA viruses
Figure 2 Application of protocol to field isolates from 2001.
Coverage of between 1000–10,000x was achieved for 4/6 UKG 2011
isolates (O/UKG/1450/2001 (blue), O/UKG/1558/2001 (green),
O/UKG/1734/2001 (purple) and O/UKG/14597/2001 (red)) with a
drop in coverage at the poly(C) tract (~375 bp position). O/UKG/
4998/2001 (orange) showed lower coverage of between 10-100x.
Primer locations are shown as black arrowheads above the
genome illustration.
protocol we replaced the type O specific primer ‘4926R’
with a pan-FMDV primer ‘NK-72’ designed to bind a region conserved between all seven FMDV serotypes
(Table 2). The panel had a viral load >1 × 108 copies/μl.
De-novo assemblies were completed to provide a
Figure 3 Genome coverage profiles for FMDV serotype panel.
Sequence data coverage at each position along the genome is
shown for serotype O (black), A (pink), Asia 1 (orange), C (green),
SAT 1 (light blue), SAT 2 (blue), and SAT 3 (yellow). The majority of
the coverage is above 1000x. In all viruses tested, a poly(C) tract
within the FMDV genome at ~375 bp was associated in a reduction
in coverage. The coverage depth observed for SAT 3 was lower than
other serotypes. Primer locations are shown as black arrowheads
above the genome illustration.
In order to demonstrate the suitability of this method to
attain whole genome sequence from other poly(A) tailed
RNA viruses, we tested the protocol upon three different
viruses including encephalomyocarditis virus 1 (EMCV-1)
equine rhinitis A virus 1 (ERAV-1) and vesicular exanthema of swine virus B34 (VESV-B34) (Figure 4). For all
three viruses, first-strand cDNA synthesis was performed
using the 3′ oligo-dT primer ‘Rev 6’ and sequence-specific
primers replacing the pan-FMDV specific NK72 (Table 2).
The complete genome sequence, apart from the poly(C)
tract was determined for EMCV-1 ATCC VR-129B
(KM269482). The complete genome sequence, apart from
100+ nt at the 5′ end of the genome was determined for
ERAV-1 D1305-03 (KM269483). Similarly, the majority of
the calicivirus VESV-B34 genome was determined apart
from six nt at the 5′ end of the genome (KM269481).
Discussion
We have described a novel sample preparation method
incorporating minimal amplification for the accurate sequencing of RNA viruses to a consensus level, using an
Illumina MiSeq. This protocol is an affordable and reproducible method to generate whole genome sequences
for FMDV and other RNA viruses, which could be
adapted to routine high-throughput diagnostic laboratory workflows. The protocol was validated using FMDV
type O (Figure 2) and shown to be applicable to all other
serotypes of FMDV (types A, C, Asia 1, SAT 1, SAT 2
and SAT 3) (Figure 3) as well as other picornaviruses
(EMCV-1 and ERAV-1) and a calicivirus (VESV-B34)
(Figure 4).
We have shown that the protocol is able to produce
whole genome sequences from samples with viral loads
as low as 1 × 107 virus RNA copies per μl. Further validation was performed with five samples submitted during
the UK 2001 FMDV outbreak. The generation of five genomes from these samples, without PCR amplification or
virus culture, demonstrated the potential for this method
to investigate larger outbreak sample sets in a highthroughput, diagnostic setting, such as the UK 2001
FMDV outbreak.
PCR processes have previously been shown to be error
prone [4] and thus eradication of this step has the opportunity to improve the quality of the data. Our protocol
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Table 2 Primers and probes used in quantitation and WGS of FMDV and other RNA viruses
RT-qPCR
First-strand synthesis
Primer name
Primer sequence
Callahan 3DF [24]
ACT GGG TTT TAC AAA CCT GTG A
Callahan 3DR [24]
GCG AGT CCT GCC ACG GA
Callahan 3DP [24]
TCC TTT GCA CGC CGT GGG AC
UKFMD Rev 6 [25]
GGC GGC CGC TTT TTT TTT TTT TTT
NK72 [26]
GAA GGG CCC AGG GTT GGA CTC
UKFMD UKG 4926R
AAG TCC TTC CCG TCG GGG T
EMC-2B65R [27]
TCG GCA GTA GGG TTT GAG
ERAV-2A22R [28]
GGG TTG CTC TCA ACA TCT CCA GCC AAT TT
Vesi-3D1R
CKN GTN GGY TTN ARN CC
Vesi-3D2R
TAN CAN CCR TCR TCN CCR TAN GT
International Union of Pure and Applied Chemistry (IUPAC) nucleotide ambiguity codes:
N: G or T or A or C; K: G or T; Y: T or C; R: G or A.
differs from previous studies in the literature through
inclusion of sequence specific primers, as opposed to random priming at the first strand cDNA stage [29,30]. This
decision was made with the intent of maximising coverage, across the whole genome, specifically for FMDV; although it is possible that primer induced bias could be
introduced into sequences through use of sequence specific primers.
We have also demonstrated the effectiveness of adapting
this method for WGS of other RNA viruses (Figure 4). We
foresee this protocol being practicable for unknown positive sense polyadenylated viruses through use of random
primers and, where appropriate, an oligo-dT primer.
Figure 4 Genome coverage profiles for three non-FMDV panel
of viruses. Coverage of 10,000 was achieved for the majority of the
EMCV-1 genome (olive). Peaks in coverage can be observed at the
location of sequence specific primers used in the RT reaction
(~4000 bp and ~8000 bp). A dip in coverage was evident at the
poly(C) tract. The ERAV-1 genome showed between 10x and 100x
coverage with visible peaks in coverage at the specific primer sites
(~4000 bp and ~8000 bp) (black). Approximately 100x coverage of
the majority of the VESV-B34 genome was achieved (blue).
The specificity previously provided by PCR has been
replaced with reduction of host DNA and the optional
use of specific primers in the reverse transcription reaction. Instead of enriching viral RNA we depleted host
genomic DNA. We did not target ribosomal RNA in
order to keep reagent costs low thus maintaining the
suitability of the protocol for ‘high-throughput’ sample
processing. The method described here was capable of
generating whole genome sequences of FMDV field
isolates with a coverage depth of up to 1 × 104 (data not
shown) that was considered sufficient for the study of
minority variants [24], with only a minimal amount of
PCR at the library preparation stage. This PCR amplification involved 10 cycles of amplification by a hi-fidelity
DNA polymerase, thereby posing minimal risk to biasing
the final sequence data [31].
It was evident that in genome sequences generated
using this protocol the genomic termini and poly(C) tract
exhibited lower coverage depths. The 5′ genomic termini
were always under-represented within the genomes. This
was particularly evident in samples of decreased viral load
suggesting that increasing the input RNA of such samples
could improve this coverage. Additionally homopolymeric
regions, such as the long poly(C) tract of FMDV, have
been demonstrated here to cause significant decreases in
coverage. With Sanger sequencing, large parts of the genome are often missing or primer derived. For example,
twenty seven to fifty nucleotides of the full genome
sequences obtained by Sanger sequencing described by
Valdazo-Gonzalez et al., [15,32-34] were primer derived
(from the forward and reverse primers to amplify 5’ and 3’
termini of both the S and the L fragment) and thus the
method described here offers a notable improvement on
the resolution of these regions. As previously stated, a
minimum read depth of coverage required to create an accurate consensus for a type O sequence was on average 22
(Figure 1). Even after implementation of this criterion,
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consensus sequences were generated with a depth of >22,
at more than 80.6% genome positions. This was observed
in the 5 UKG field isolates tested and >99.6% for type O
virus tested as part of the panel of serotypes (Table 1).
Such advances in WGS will likely impact fields such as
virus evolution, diagnosis, and generation of high/low
pathogenicity variants. We have already shown this
method can be advantageous in a diagnostic setting with
the successful sequencing of 94.1% of the genome of a
culture negative field isolate. FMDV reads were successfully identified although the resulting profile exhibits
several gaps in the genome sequence suggesting that the
RNA was in fact degraded - an observation potentially
explaining the inability for this virus to grow successfully
in cell culture. For this protocol to be fully functional
within a diagnostic environment, it remains to be confirmed whether it is able to correctly identify all viruses
or serotypes within mixed samples.
Conclusion
This paper outlines the development of a high-throughput
protocol for the generation of whole genome sequences of
all seven serotypes of FMDV. With minimal changes applied to priming in the first strand synthesis stage such a
strategy can be tailored to other RNA viruses. The application of NGS to virology will prove invaluable to the fields
of molecular epidemiology and phylogenetic outbreak
tracing. This paper describes a fast, robust and affordable
protocol, which is essential to realise this potential.
Methods
Virus specimens
The protocol was initially developed and validated using
an FMDV field isolate (O/UKG/35/2001) submitted to the
FAO World Reference Laboratory for FMD (WRLFMD,
Pirbright, UK) during the 2001 FMD outbreak in the
United Kingdom. It was further validated with a panel of
other samples originating from this outbreak as well as
with a panel of viruses representing all FMDV serotypes.
The protocol was also validated with other representative
polyadenylated RNA viruses. The details of all viruses used
in the study are described in Table 3. Where appropriate,
viruses were cultured for one replication cycle in bovine
thyroid cells (BTy) as described previously [35]. Dilutions
between 1 × 108 to 1 × 106 viral copies/μl of O/UKG/35/
2001 were made with viral cell culture supernatant in virus
negative suspensions of bovine epithelium to mimic real
clinical samples with different viral loads.
RNA extraction & FMDV-specific RT-qPCR
Total RNA was extracted from 460 μl of cell culture
virus isolate or original suspension [consisting of 10%
tissue suspensions generated in M25 phosphate buffer
(35 mM Na2HPO4.2H2O; 5.7 mM KH2PO4; pH 7.6;
Table 3 Viruses used in development and validation of the non-amplification protocol
Family
Genus
Picornaviridae Aphthovirus
Caliciviridae
Species
Serotype
Foot-and-mouth disease virus
O
Isolate
Passage history (Cell type/Passage
number)
UKG/1734/2001
10% epith. susp.
UKG/1450/2001
10% epith. susp.
UKG/14597/2001
10% epith. susp.
UKG/1558/2001
10% epith. susp.
UKG/4998/2001
10% epith. susp.
UKG/1485/2001
10% epith. susp.
UKG/35/2001
10% epith. susp.
TUR/11/2013
BTy2
A
TUR/12/2013
BTy2
C
KEN/1/2004
BTy2
Asia 1
TUR/13/2013
BTy2
SAT 1
TAN/22/2012
BTy2
SAT 2
TAN/5/2012
BTy2
SAT 3
ZIM/6/91
BTy2
Equine rhinitis A virus
1
D1305-03, dromedary, Dubai,
2003
Vero2
Cardiovirus
Encephalomyocarditis virus
1
VR-129B, chimpanzee, Florida,
1944
BHK3
Vesivirus
Vesicular exanthema of swine
virus
B34
B1-34, pig, California, 1934
PK5, IB-RS5
BTy: Primary Bovine Thyroid; PK: Pig kidney epithelial cells; BHK: Baby Hamster Kidney; IB-RS: instituto Brazilia Renal Swine; Numbers denote passage number.
Logan et al. BMC Genomics 2014, 15:828
http://www.biomedcentral.com/1471-2164/15/828
made in-house)] using RNeasy MiniKit (Qiagen) according to manufacturer’s instructions. Total RNA was eluted
in 50 μl of nuclease-free water and quantified using the
Qubit RNA High Sensitivity (HS) Assay Kit (Life Technologies). FMDV-specific RNA was detected using an
FMDV-specific real-time RT-qPCR as described previously (Table 2) [24] and quantified using an RNA standard
derived from O/UKG/35/2001.
gDNA depletion
Genomic DNA (gDNA) was depleted from extracted total
RNA samples through the activity of rDNase1 using the
DNA-free DNAse kit (Life Technologies). Briefly, 10 μg of
extracted nucleic acid in a 50 μl volume was combined
with 5 μl of DNase Buffer and 1 μl of rRDNase1 (2 U),
and incubated at 37°C for 30 min. Inactivation agent was
added as per manufacturer’s protocol and the sample was
incubated for a further 2 min at room temperature with
periodic mixing. The samples were then centrifuged at
17,000 xg for 2 min and the DNAse-treated supernatant
was retained for subsequent processing.
cDNA synthesis
First-strand cDNA synthesis (reverse transcription) was
performed using Superscript III First-Strand Synthesis System (Life Technologies) according to the manufacturer’s
protocol. Briefly, 10 μl of DNase-treated total RNA was
combined with oligonucleotide primers (Rev6 (2 μM),
NK72 (2 μM) or FMDV-4926R (2 μM)) depending on the
application of the protocol, random hexamers (50 ng/μl:
Life technologies), dNTPS (10 mM: Life Technologies)
and nuclease-free water (Life Technologies) (Table 2). Reactions were incubated at 65°C for 5 min and cooled on
ice for 5 min. A second reagent mix was added containing
SuperScript III enzyme (200 U: Life Technologies), RNaseOUT (40 U: Life Technologies), 0.1 M dTT (life Technologies) and 25 mM MgCl2, before incubating at 50°C for
50 min. A final incubation with RNase H (2 U: Life Technologies) was then performed at 37°C for 20 min.
Second-strand synthesis was performed using NEB Second Strand Synthesis kit (NEB) as per manufacturer’s instructions using 20 μl of cDNA. The resulting dsDNA was
purified using Illustra GFX DNA/gel clean-up kit (GE) as
per manufacturer’s instructions and samples eluted in
30 μl of nuclease-free water. Double-stranded cDNA samples were then quantified using the Qubit dsDNA High
Sensitivity (HS) Qubit kit (Life Technologies) after which
samples were adjusted to 0.2 ng/μl using nuclease-free
water where appropriate prior to library preparation.
Illumina library preparation
One nanogram of each dsDNA sample was used to prepare sequencing libraries using the Nextera XT DNA Sample Preparation Kit (Illumina) according to manufacturer’s
Page 8 of 10
instructions. Libraries were sequenced on a MiSeq using
300 cycle version 2 reagent cartridges (Illumina) to produce paired end reads of approximately 150 bp each.
Sequence data analysis
Consensus sequences were attained using a complete published sequence as a template or, where a closely related
template was not available, a de novo assembly. Sequence
read quality was monitored with FastQC [36] prior to
Sickle [37] trimming all bases with a q score of <30. For
de novo trimmed Fastq files were processed using Velvet
v1.2.10 [38] with an optimum Kmer length determined by
Velvet-Optimiser. A minimum contig length of 1000 was
included in L fragment analysis. A BLAST search with the
contigs confirmed viral origin [39]. Final contig assemblies
were completed manually in BioEdit [40]. Alignments
between MiSeq data and appropriate reference genome
(from publication or de novo assembly) were completed
using Bowtie2.1.0 [41] and SAM/BAM processing carried
out using Samtools [42]. Alignments were visually checked
using Tablet [43]. Coverage data and graphs were generated using Bedtools [44] with final graphical output produced using Prism v6 (GraphPad).
Sequence data deposition
All genome sequences produced in this study were submitted to NCBI GenBank under the following accession
numbers.
UK2001 FMDV field isolates
O/UKG/1450/2001 [KM257061], O/UKG/1558/2001
[KM257062], O/UKG/1734/2001 [KM257063], O/UKG/
14597/2001 [KM257065] and O/UKG/4998/2001 [KM2
57064].
Different FMDV serotypes isolates
O/TUR/12/2013 [KM268895], A/TUR/11/2013 [KM26
8896], C/KEN/1/2004 [KM268897], Asia1/TUR/13/2013
[KM268898], SAT1/TAN/22/2012 [KM268899], SAT2/
TAN/5/2012 [KM268900] and SAT3/ZIM/6/91 [KM26
8901].
Non-FMDV viruses
VESV-B34 [KM269481], EMCV-1 VR-129B [KM269482]
and ERAV-1 D1305-03 [KM269483].
Read coverage required to obtain an accurate consensus
sequence
A sorted alignment file (.sam) of FMDV O/UKG/35/2001
was generated using Bowtie2.1.0 [45] and Samtools [42]. A
bespoke python script that truncated the samtools mpileup output format (available upon request) was used to
simulate files with varying levels of coverage. A consensus
sequence was generated from each of these files using
Logan et al. BMC Genomics 2014, 15:828
http://www.biomedcentral.com/1471-2164/15/828
mpileup (Samtools). The consensus sequences created
were compared in BioEdit [40] and their sequence identities recorded. This was completed for all FMDV type O
isolates with a sufficient number of reads and the mean
was calculated.
Page 9 of 10
3.
4.
5.
Ethics
Our animal use protocols conform to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines [46] for reporting animal studies. All samples were
collected with the informed institutional and client consent under the highest standards of veterinary care.
Additional file
Additional file 1: Figure S1. A. Genome coverage profile for FMDV/O/
ISR/2/2013. The Israel 2013 isolate of FMDV O was negative when tested
in cell culture in IB-RS-2 and BTy cells. This protocol provided coverage of
above 10x for the majority of the genome although full genome consensus
was not acquired. The expected dip in coverage at the poly(C) was observed.
Primer locations are shown as black arrowheads above the genome
illustration.
6.
7.
8.
9.
10.
Abbreviations
NGS: Next generation sequencing; WGS: Whole genome sequencing;
FMDV: Foot-and-mouth disease virus; FMD: Foot-and-mouth disease; PCR
(RT-qPCR): Reverse transcription-quantitative; EMCV-1: Encephalomyocarditis
virus 1; ERAV-1: Equine rhinitis A virus 1; VESV-B34: Vesicular exanthema of
swine virus B34; WRLFMD: World Reference Laboratory for FMD;
UTR: Untranslated region; VP1: Viral protein 1; gDNA: Genomic DNA;
BTy: Bovine thyroid cells; PK: Pig kidney epithelial cells; BHK: Baby hamster
kidney cells.
Competing interests
The authors declare that they have no competing interests.
11.
12.
13.
14.
Authors’ contributions
GL, GF and EMC devised the protocol, performed the experimental work,
performed the analysis and wrote the paper. DJK, KB and BVG carried out
additional experiments and analysis; NS assisted in analysis, NJK provided
samples and assisted in analysis, DPK was involved in study design and
writing the paper. All authors read and approved the final manuscript.
15.
Authors’ information
Grace Logan and Graham L. Freimanis are joint first authors.
16.
Acknowledgements
This study was funded by a PhD studentship supported by The Pirbright
Institute and the University of Glasgow, and research projects from
EMIDA-ERA (EpiSeq), BBSRC (BB/I014314/1) and the UK Department for
Environment, Food and Rural Affairs (Defra: SE2940). DPK, NJK and EC are
partially supported by an ISPG provided by the BBSRC. The authors
acknowledge support from colleagues within the FMD Reference Laboratory
at Pirbright for providing the samples used in this study.
17.
Received: 6 May 2014 Accepted: 22 September 2014
Published: 30 September 2014
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Cite this article as: Logan et al.: A universal protocol to generate
consensus level genome sequences for foot-and-mouth disease virus
and other positive-sense polyadenylated RNA viruses using the Illumina
MiSeq. BMC Genomics 2014 15:828.
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